| AVS 54th International Symposium | |
| Biomaterial Interfaces | Wednesday Sessions |
| Session BI-WeA |
| Session: | Nucleic Acid Sequencing and Technology |
| Presenter: | P.-C. Nguyen, University of Washington |
| Authors: | P.-C. Nguyen, University of Washington L.J. Gamble, University of Washington C.-Y. Lee, 3M Corporate Research Analytical Laboratory G.M. Harbers, University of Utah B.J. Tyler, University of Utah D.W. Grainger, University of Utah D.G. Castner, University of Washington |
| Correspondent: | Click to Email |
The printing process for preparing DNA microarrays introduces variability in microspots, as observed with fluorescence detection commonly used to analyze these arrays. The nanolitre drops of solution printed onto the microarray surface dries within seconds. During this rapid drying the solution ionic strength and solute concentrations increase dramatically. Interspot variations and non-uniform distribution of probe molecules within spots are major sources of experimental uncertainty in microarray analysis. Various primary ion sources (Bi+, Bi3+, Bi3++, C60+, C60++ and C60+++) were used in imaging time-of-flight secondary ion mass spectrometry (ToF-SIMS) to study this non-uniformity. The type of information gained from using different primary ion beams is compared. Principal component analysis (PCA) and maximum autocorrelation factors (MAF) were used to analyze the image results and determine which masses were the main causes of the observed variability. Amine-modified single-stranded DNA was immobilized on commercial slides containing NHS groups. Spots containing 0 to 100% of fluorescent Cy3 labeled DNA were examined. Different percentages of Cy3 label resulted in variations in spot size and shape as well as differences in fluorescence distribution within spots. Imaging ToF-SIMS showed that additives in the print solution (sodium dodecyl sulfate, N-lauroyl sarcosine, salts, etc.) as well as the Cy3 labeled DNA were non-uniformly distributed within the microspots. These non-uniformities were more apparent in images acquired with Bi3+ and Bi3++ compared to images acquired with Bi+. Compared to univariate analysis (i.e., examination of individual masses), both PCA and MAF methods more readily highlighted the distributions of chemical non-uniformities present in the DNA microspots. Results thus far indicate that most detail about types and distribution of chemical species in DNA microspots have been obtained from MAF analysis of the Bi3++ images. However, C60++, C60+++ data is providing additional information that is been examined.